Gut Microbiota Shape Oligodendrocyte Response after Traumatic Brain Injury

White matter injury (WMI) is thought to be a major contributor to long-term cognitive dysfunctions after traumatic brain injury (TBI). This damage occurs partly due to apoptotic death of oligodendrocyte lineage cells (OLCs) after the injury, triggered directly by the trauma or in response to degenerating axons. Recent research suggests that the gut microbiota modulates the inflammatory response through the modulation of peripheral immune cell infiltration after TBI. Additionally, T-cells directly impact OLCs differentiation and proliferation. Therefore, we hypothesized that the gut microbiota plays a critical role in regulating the OLC response to WMI influencing T-cells differentiation and activation. Gut microbial depletion early after TBI chronically reduced re-myelination, acutely decreased OLCs proliferation, and was associated with increased myelin debris accumulation. Surprisingly, the absence of T-cells in gut microbiota depleted mice restored OLC proliferation and remyelination after TBI. OLCs co-cultured with T-cells derived from gut microbiota depleted mice resulted in impaired proliferation and increased expression of MHC-II compared with T cells from control-injured mice. Furthermore, MHC-II expression in OLCs appears to be linked to impaired proliferation under gut microbiota depletion and TBI conditions. Collectively our data indicates that depletion of the gut microbiota after TBI impaired remyelination, reduced OLCs proliferation with concomitantly increased OLC MHCII expression and required the presence of T cells. This data suggests that T cells are an important mechanistic link by which the gut microbiota modulate the oligodendrocyte response and white matter recovery after TBI.


Introduction
In the United States, approximately 1.7 million people experience traumatic brain injury (TBI) each year, and over 5 million face TBI-related disabilities (1).The overall health cost attributable to nonfatal TBI in the United States has been estimated to be over $40 billion per year (2).Traumatic white matter injury (WMI) is thought to be a major contributor to long-term cognitive dysfunction in TBI survivors (3)(4)(5).Oligodendrocytes, which provide metabolic support to axons and are the producers of myelin in the central nervous system (CNS), undergo apoptosis after TBI, triggered by direct injury or in response to axonal degeneration (6, 7).Mature oligodendrocytes present in the brain at the time of injury have limited, if any, capability to contribute to remyelination, with the bulk of CNS remyelination attributed to new oligodendrocyte production by oligodendrocyte lineage cells (OLCs) (8-10).OLC-mediated remyelination involves a three-phase process of proliferation, recruitment, and differentiation, which can be adversely impacted by in ammation after TBI (11,12).
Research over the past few years has revealed that the gut microbiota in uence neurogenesis, myelination and subsequently functional and behavior outcomes (13,14).This complex interplay is known as the gut-brain axis, a bidirectional communication that includes neuro-endocrine-immunological activity (15).Early-life gut microbial depletion has been shown to have long-term effects on maturation of myelinating oligodendrocytes leading to altered cognition and anxiety-like behavior (16).The gut microbiota has been shown to modulate the in ammatory response after acute CNS injury impacting injury severity and recovery (17)(18)(19).Recently, we showed that gut microbiota depletion after TBI alters the innate and adaptive immune response (Celorrio et al., 2021).The innate immune response plays an important role in myelin debris clearance which in uences OLC proliferation and migration (20).Activated T-cells have been found to have a trophic role by promoting OLC proliferation in vitro (21), while effector T-cells inhibit OLCs differentiation in an in ammatory demyelinating mouse model (Kirbi et al., 2019).
However, the possible T cell-OLC crosstalk and the mechanism of cell-cell communication after WMI hasn't yet been elucidated.Gut microbiota perturbations are common after TBI (22,23), but their potential impact on remyelination after traumatic WMI is currently unknown.Therefore, we hypothesized that the gut microbiota plays a critical role regulating the OLC response to traumatic WMI in uencing T-cells differentiation and activation.We evaluated whether an intact enteric microbiome early after TBI is critical for OLC proliferation and remyelination.We further investigated if the presence of T cells is required for gut microbial modulation of the OLC response and subsequent remyelination following TBI.Finally, using an in vitro model system we further explored OLC/T-cell interactions and the role of the gut microbiome.

Animals
All procedures were approved by the Washington University Animal Studies Committee (Protocol 19-0864) and are consistent with the National Institutes of Health guidelines for the care and use of animals.Animals were housed 5/cage and had free access to water and food with a 12-hour light/dark cycle.C57BL/6J (RRID: IMSR_ORNL:C57BL/6J-A/A) and B6.129P2-Tcrb tm1Mom Tcrd tm1Mom/J J (TCRb -/- TCRd -/-) (RRID:IMSR_JAX:002122) 8 week-old male and female mice (Jackson Laboratory, Bar Harbor, ME) weighing 20-25 grams (g) were used for in vivo studies.C57BL/6J male and female neonatal P2-P5 pups were used for in vitro studies.

Controlled cortical impact with gut microbial depletion
Controlled cortical impact (CCI) was performed using a previously described protocol (17).Mice were anesthetized with 5% iso urane and maintained at 2% iso urane throughout the procedure.
Buprenorphine sustained-release (0.5 mg/kg) was administered subcutaneously, prior to scalp incision.Ear bars were positioned to secure the head within the stereotaxic frame (MyNeurolab, St. Louis, MO).A 5 mm craniectomy was performed using an electric drill, centered 2.7 mm lateral to the midline and 3 mm anterior to lambda.Animals were randomly assigned to either CCI or sham group after craniectomy using a computer-generated randomization algorithm.The electronic impactor (Leica Biosystems, Richmond, VA) equipped with a 3 mm tip was aligned with the craniectomy site using the following coordinates: 1.2 mm lateral to the midline and 1.5 mm anterior to lambda (Celorrio et al., 2021).The impact was delivered at a depth of 2 mm with a velocity of 5 m/s and a dwell time of 100 ms.A loose-tting 7 mm plastic cap was secured over the craniectomy site using Vetbond (3M, St. Paul, MN).The skin incision was closed with interrupted sutures and treated with antibiotic ointment.Animals were then placed on a warming pad for recovery.
For gut microbiota depletion, broad-spectrum antibiotics were administered for 7 consecutive days in the drinking water consisting of 250 mg vancomycin, 500 mg neomycin sulfate, 500 mg ampicillin, 500 mg metronidazole (VNAM), and 10 g grape-avored Kool-Aid (Kraft Heinz, IL, Chicago) in 500 mL of sterileltered water (Steed et al., 2017).Control animals received only Kool-Aid in drinking water to reduce the bitter avor of the antibiotics.

Fecal microbiota transplantation
We performed two fecal microbiota transplantations (FMTs) of the gut microbiota from VNAM-or Kool-Aid-treated uninjured animals into germ-free (GF) mice as previously described (24).Brie y at Day 17 and Day 10 prior to injury, GF mice received FMTs from animals with 7 days of VNAM or Kool-Aid treatment.
For each extraction, fecal pellets were collected 7 days after initiation of VNAM or Kool-Aid treatment.The pellets were mixed with phosphate-buffered saline (PBS) and after 5 min, the sample was vortexed to break up the fecal matter and allowed to sit for ∼5 min to allow for debris to settle.The supernatant was then removed with an uncut P1000 pipette tip into a sterile 5 mL tube to avoid material clogging the gavage needle.Mice were gavaged with a sample volume of 250 μL.Ten days after the last FMT, we performed CCI in all the mice and they remained in sealed cages in the animal facility for 7 days before being euthanized.

Tissue Processing
Mice were euthanized under iso urane anesthesia, by transcardial perfusion with cold 0.3% heparin in PBS followed by 4% paraformaldehyde solution in PBS (PFA, Sigma-Aldrich, St. Louis, MO).Brains were post-xed in 4% PFA for 24 h at 4 °C followed by equilibration in 30% sucrose for 48 h before sectioning.Using a freezing microtome, brains were cut, and four 50-μm thick cryosections with complete CC spaced 300 μm apart were used for the subsequent analysis.
Myelin Black Gold II staining Myelin Black Gold II (BGII, Histo-Chem, Jefferson, AR) staining was performed as previously described (Celorrio et al., 2022;Shumilov et al., 2023).Brie y, to visualize individual myelin bers in the CC and quantify myelinated percent area, BGII staining was performed on four 50-μm thick slices spaced 300 μm apart with the most rostral slice being the rst appearance of the dorsal hippocampus.Free-oating slices were rinsed 3 times with tris-buffered saline (TBS) for 5 min at room temperature (RT), and then incubated for 12 min at 60°C in pre-warmed BGII solution (0.3% in 0.9% NaCl), followed by 2 washes in distilled water at RT. Next, slices were incubated in pre-heated sodium thiosulfate (1% in distilled water) at 60°C for 3 min.After 3 washes in TBS, tissue was mounted on charged slides and dried overnight.Slides were dehydrated using a serial of graded alcohols (50%, 70%, 95%, and twice with 100%) and coverslipped with DPX (Sigma-Aldrich, St. Louis, MO).Images were generated using 20X objective with a Bright eld Zeiss Axio Scan Z1 microscope (Zeiss, White Plains, NY).Percent of myelinated area of the CC was quanti ed using ImageJ software (25).The CC region of interest was de ned as the area between the mid CC and the cingulum unless it was truncated by the injury.

Fluorescence Immunohistochemistry
Fluorescence immunohistochemical staining was performed on free-oating sections.Tissue was incubated with pre-heated HCl 1N (Sigma-Aldrich) for 30 min at 45°C to increase the antigen exposure for BrdU detection.After the three washes with PBS, 20% normal donkey serum, 3% bovine serum albumin, and 0.3% triton X-100 in PBS were used to block nonspeci c staining for all antibodies.
Sections with a thickness of 50 μm were stained with the primary antibodies (Table 1) at 4°C overnight.The next day, antibody binding was detected by incubating sections with Alexa Fluor secondary antibody (Table 1) for 2 hours in PBS with 0.3% triton X-100.Sections were mounted on glass slides in PBS, dried, and coverslipped with mounting medium for uorescence with 4',6-Diamidino-2-Phenylindole, (DAPI, Thermo Fisher Scienti c, Waltham, MA).

Quantitative uorescent immunohistochemistry
Fluorescent images were obtained with a Zeiss Axio Imager Z2 with ApoTome 2 uorescence microscope with a 20X objective.20-µm z stacks with an interval of 1 µm were obtained of the ipsilateral CC.Quanti cation of OLC proliferation was performed by counting the number of cells that co-localized with nuclear and/or cytoplasmic Olig1 immunolabeling and BrdU staining in 4 slices spaced 300 µm apart by a blinded observer.Degraded myelin basic protein (dMBP) uorescent immunostaining was performed on adjacent sections.Images were generated using 20X objective with a uorescence slide scanner Zeiss Axio Scan 7 microscope (Zeiss, White Plains, NY).dMBP percent area of the CC was quanti ed using ImageJ software (25).

T-cells isolation from spleen
Spleens were collected from injured animals treated with VNAM or Kool-Aid as described above.Splenocytes were obtained by mechanical shredding and ltered through a 70-µm cell strainer, and centrifuged at 500g for 10 min.The resulting cell suspensions were incubated with red blood lysis buffer (Roche Diagnostics Gmbh, Mannheim, Germany) for 5 min at 4°C, centrifuged and ltered through 40-µm cell strainer.T-cells were then isolated following manufacturers protocol by negative selection using pan T-cell isolation kit II (Miltenyi biotec).This isolation kit is based on a cocktail of biotin-conjugated antibodies against CD11b, CD11c, CD19, CD45, CD49b, CD105, Anti-MHC-class II, and Ter-119.Cells were counted and co-cultured with OLCs at a density of 3x10 4 cells/well on a 96-well plate or 3x10 5 cells/well on a 6-well plate at 37°C and 5% CO 2 for 24 h.Immunocytochemistry Cells were permeabilized using 0.3% TX-100 in PBS for 10 min.Then cells were incubated with HCl 1N (Sigma-Aldrich, St. Louis, MO) for 30 min at 45°C to increase the antigen exposure for BrdU detection.Non-speci c antibody interactions were blocked using 20% NDS in PBS for 1 h.Primary antibodies (Table 1) were diluted in the same blocking solution and incubated overnight at 4°C.The cells were washed with PBS and secondary antibodies (Table 1) diluted in PBS were incubated for 2 h.For nuclei detection, cells were incubated for 10 min with DAPI (1:5000, Life Technology, Carlsbad, CA) and stored at 4°C in PBS.The immuno uorescent images were taken using Zeiss Celldiscover 7 (Zeiss, White Plains, NY) with 10x objective, 4 tiles per well were automatically scanned.We used ImageJ (NIH public software) particle analysis plugin with macro instructions (27) to quantify the number of cells and percent of area immunostained.To analyze the co-localization of BrdU and DAPI positive cells quantitative analysis of Mander's coe cient were performed using the JACoP plugin for ImageJ.

Flow cytometry analysis
After 24 h of co-culture with OPC, T-cells were stimulated for intracellular cytokines expression in vitro.Supernatant from the co-culture were collected centrifuged at 300 g for 1 min and resuspended in RPMI 1640 with glutamax, 10% FCS, 12.5 mM of Hepes (Gibco), 1% of Pen/Strep (Gibco, Waltham, MA), 50µM of B-mercaptoetanol (Sigma-Aldrich, St. Louis, MO) and 10µg/ml of gentamycin (Sigma-Aldrich) with 100 ng/ml of phorbol 12-myristate 13-acetate (PMA), 1 µg/ml of ionomycin (Sigma-Aldrich) and 1x brefeldin (BioLegend, San Diego, CA) at 37°C and 5% CO 2 for 4 h.OPCs were detached from the surface of the culture plate by repetitive resuspensions in FACS buffer and collected in 1 ml tubes, followed by centrifugation at 300 g for 7 min.Next, cells were incubated for 5 min with Zombie NIR Dye (BioLegend, San Diego, CA).Then, cells were washed with FACS buffer, stained with their respective antibody mix (Table 1) for 30 min at RT, and analyzed on a BD LSRFortessa ow cytometer (BD Biosciences, Franklin Lakes, NJ) using the Software v10.6.1 (BD Biosciences, Franklin Lakes, NJ).T-cells were de ned as

Statistical analysis
Blinding of investigators to experimental groups was maintained until data were fully analyzed.Data were assessed for normal distribution with the Shapiro-Wilk test and expressed as mean ±SEM.Twotailed Student's t-test was used when comparing two conditions.For more than two conditions, ANOVA and Tukey's multiple comparison post-hoc test were employed.All analysis was performed with GraphPad Prism v10.1.0(GraphPad software.Boston, MA).

Results
Gut microbiota depletion impedes white matter repair three months after TBI Our previous work has demonstrated that gut microbial depletion for 1 week signi cantly impacts neuroin ammation and fear memory 3 months after TBI (17).Since, chronic white matter degeneration is in uenced by prolongated neuroin ammation (28), we decided to investigate the impact of gut microbiota depletion on white matter remyelination 3 months after TBI.Gut microbiota was depleted by administering broad-spectrum antibiotics orally to mice immediately after CCI for 1 week (Fig. 1a).Three months post-TBI, white matter remyelination was assessed by staining the peri-contusional CC with BGII and measuring the percentage of myelinated area adjacent to the lesion site (Fig. 1b and c).Our ndings revealed that early gut microbiota depletion after injury signi cantly reduced the percentage of myelinated area in the CC compared with Kool-Aid-treated animals 3 months post-TBI (Fig. 1c).However, the underlying mechanisms by which gut microbiota in uences post-TBI recovery remain to be elucidated.Therefore, we next performed a more in-depth analysis of gut microbiota depletion impact on acute WMI.Depletion of gut microbiota decreases oligodendrocyte lineage cell proliferation and increases myelin debris one week after TBI Next, we wanted to further explore the effect of gut microbiota depletion on acute WMI and remyelination 7-day post-TBI (Fig. 2a).One critical inhibitor of remyelination and oligodendrocytes proliferation is myelin debris accumulation (29).After WMI, myelin debris is accumulated specially in CC due to glial dysfunction and can be associated with axon regeneration impairment and neuroin ammation (30).We found an increase in myelin debris accumulation in the CC of VNAM-treated injured mice compared with injured controls (Fig. 2b-c).Additionally, gut microbiota depletion suppressed OLC proliferation, a crucial step in remyelination, as indicated by a signi cant reduction in BrdU/Olig1 double-positive cells (Fig. 2de).The density of b-APP swelling, indicative of axonal injury, did not differ between VNAM and Kool-Aid treated mice one week after TBI (Fig. 2f-g).Furthermore, to determine if the impact of gut microbiota depletion on the white matter remyelination after TBI was mediated directly by antibiotics or indirectly via modulation of the gut microbiota, we utilized a fecal microbiota transplantation (FMT) approach (Fig. 2h).Consistent with the results described above, GF mice that received FMT from VNAM-treated animals exhibited signi cantly higher percent area of myelin debris accumulation compared to those receiving Kool-Aid FMT (Fig. 2i-j).Taken together, these results support our hypothesis that the gut microbiota plays a pivotal role in modulating post-traumatic myelin debris clearance and OLC proliferation.

Pharmacological depletion or genetic deletion of T-cells rescues OLC proliferation and remyelination impaired by gut microbial depletion
Previously, we have demonstrated that gut microbiota depletion after TBI impairs hippocampal neurogenesis, promotes and pro-in ammatory microglia phenotype and surprisingly reduced T cell in ltration into the brain (17).Building upon these ndings, we wanted to further investigate the role of the T-cells in gut-brain communication in the context of WMI.To achieve the depletion of T-cells, we employed pharmacological treatment with anti-CD3 IgG for one month, to be able to address changes in myelin density (Fig. 3a).Peripheral blood analysis following T-cell depletion revealed a nearly complete absence of CD4 + (Fig. 3b) and CD8 + (Fig. 3c) lymphocytes.Surprisingly, the percent of myelinated area (Fig. 3d and f) and impaired OLC proliferation (Fig. 3e and g), effect of gut microbial disruption, was restored after the depletion of T-cells compared to VNAM-treated mice with control IgG injections.
Furthermore, TCRb -/-TCRd -/-mice (absence of alpha beta T-cell receptor and any gamma delta T-cell receptor) exposed to VNAM for one week (Fig. 4a) showed similar ndings.The density of oligodendrocyte lineage proliferative cells (Fig. 4b-c), and myelin debris accumulation (Fig. 4d-e) remained unaffected by microbial depletion in TCRb -/-TCRd -/-mice.Taken together, these ndings suggest that T-cells have a crucial role in gut-brain communication and the modulation of remyelination following traumatic WMI.
In vitro co-culture of T-cells derived from injured mice with gut microbiota depletion reduced proliferation of OLCs OLCs are susceptible to damage from in ammatory environments triggered by T-cell cytokine production (Larochelle et al., 2021).This exposure leads to a signi cant shift in their gene expression pro le (Falcao et al., 2018).Cytokines, such as IL17 further modulate OLC functions, leading to apoptotic death of oligodendrocytes (31).Since, the absence of T-cells mitigated the impact of gut microbiota depletion on traumatic WMI repair, we decided to investigate the mechanism of interaction between OLCs and T-cells in vitro (Fig. 5a).T-cells isolated from spleens of injured animals with and without microbiota depletion 7 days after injury, were co-cultured with OLCs.We found decreased Olig1 + (Fig. 5c), and BrdU + (Fig. 5d) cell density when OLCs were co-cultured with T-cells from injured mice with microbiota depletion.Analysis using Mander's coe cient revealed co-localization of 96% of BrdU staining with Olig1 (0.9606 ± 0.02), indicating that the vast majority of proliferating cells were OLCs.Additionally, morphological analysis of OLCs showed an increase of circularity coe cient, indicative of a decreased maturation (32), when T-cells derived from injured animals with gut microbiota depletion were added to the medium (Fig. 5e).To further explore this cellular interaction, medium from T-cell and OLCs culture where exchanged (Sup.Fig. 1a), however, no changes of Olig1 or BrdU density was detected after 24h of culture (Sup.Fig. 1b-c).
Next, we characterized the T-cell differentiation from the in vitro co-culture using ow cytometry (Fig. 6a).No signi cant differences were observed in the total number of CD3 + lymphocytes (Fig. 6b).
However, the percentage of CD8 + (Fig. 6c) were signi cantly lower in both co-culture groups, and CD4 + (Fig. 6e) showed a decrease only in the Kool-Aid group.The percentage of CD4 + T-cells expressing IL17 (Fig. 6f) cytokines was signi cantly higher in lymphocytes derived from spleens of injured mice with depleted gut microbiota compared with injured controls.No signi cative changes were detected when IL4 (Fig. 6g) expression was analyzed.Furthermore, when OPCs medium was added to T-cells culture a similar increase IL17 (Sup.Fig. 1g) was detected in T cells derived from gut microbiota depleted mice.Collectively, our ndings provide evidence that differentiation of T-cells towards more pro-in ammatory phenotype could impair white matter repair after TBI through the modulation of OLC proliferation.
To further elucidate the nature of T-cell and OLC interactions in vitro, we employed a trans-well co-culture system (Fig. 7a).The permeable barrier allowed media exchange while preventing direct cell contact.Interestingly, Olig1 + (Fig. 7c), and BrdU + (Fig. 7d) presented a trend towards a decreased OLCs density.However, CD4 + lymphocytes derived from spleens of VNAM treated and injured animals exhibited signi cantly elevated expression of IL17 (Fig. 7f) cytokine compared to Kool-Aid controls.This nding suggests that, while modulation of OLC proliferation could have a greater impact when contact with Tcells is direct, CD4 + lymphocytes differentiation occurs indirectly through extracellular signaling.Our ndings provide additional evidences of the immunomodulatory role of OLCs, highlighting their complex bidirectional interplay with T-cells.

Gut Microbiota depletion increase T-cell-induced MHCII expression in OLCs after TBI
There is a growing body of evidence suggesting that OLCs can express immunomodulatory factors such as cytokines/chemokines and their receptors (33, 34) et al., 2014).T-cells/OLCs bidirectional communication was described in a previous study, where OLCs increased expression of immunoprotective genes suggesting a potential mechanism of immune functions in the context of multiple sclerosis (MS) disease (35).To further explore the mechanism of bidirectional communication between T-cells and OLCs, we decided to analyze major histocompatibility complex II (MHC-II) expression.The presence of genes associated with MHC class I and II was found in OLCs in response to interferon exposure (35,36).OLCs MHC-II intensity values was analyzed in vitro, co-cultured with direct T-cells contact (Fig. 8a), and with trans-wells cell culture (Fig. 8b).Additionally, Olig1 + /MHC-II + cell density was analyzed in the peri-contusional CC (Fig. 8c).We found that OLCs exposed to T-cell derived from microbiota depleted animals had a signi cantly higher MHC-II intensity mean value compared with control (Fig. 8d).Surprisingly, this difference vanished under in vitro trans-well co-culture conditions (Fig. 8e), suggesting that cell-to-cell contact might be necessary for T-cell-mediated MHC-II expression on OLCs.Furthermore, Olig1 and MHC-II co-localization signi cantly increased in peri-contusional CC of injured and VNAM-exposed animals compared with injured controls (Fig. 8f), indicating potential activation of immunomodulatory functions in OLCs.This data supports the potential immunomodulatory functions of OLCs under gut-microbial depleted context and the pivotal role of T cells in gut-brain communication.

Discussion
This report provides evidence that the gut-brain axis in uences white matter remyelination after TBI.Depletion of the gut microbiota after TBI impaired OLC proliferation and resulted in long-term reductions in white matter remyelination.Furthermore, our data suggests that T-cells play an important mechanistic link in gut-brain communication in regards to OLC proliferation and remyelination.Consistent with observations of MS related disease, where MHC-II expressing OLCs could activate effector T-cells (35), we show that T-cells from a gut microbial depleted host could differentiate into IL17 CD4 + in presence of OLCs in vitro.While MHC-II expression in OLCs appears to be linked to impaired proliferation.Collectively, our ndings suggest that oligodendrocytes are not passive in the neuroin ammatory and degenerative environment caused by brain trauma but instead could exert an active role in the modulation of immune response.
Extensive research has established the microbiome's in uence on in ammatory responses and immunology (37)(38)(39).Notably, its role in shaping brain development and regulating central nervous system (CNS) functions is becoming increasingly evident (40).Disruption of gut microbial composition and diversity has been linked to various neurodevelopmental disorders, including autism spectrum disorders, depression, and schizophrenia (38,41,42).Furthermore, the gut microbiome has the potential to modulate white matter structural integrity in a diet-dependent manner (43).Our investigation into TBI and gut microbial depletion reveals a profound impact on WMI recovery.We observed impaired white matter remyelination, characterized by decreased OLC proliferation and myelin debris accumulation, following TBI in animals with depleted microbiota.This correlation was further demonstrated through microbial transplantation in GF mice, showing a direct impact of the gut microbiota on WMI.This evidence emphasizes the gut microbiota's potential as a modulator of post-TBI recovery.TBI triggers complex immune responses, including the recruitment of lymphocytes to the injured site.While T-cell in ltration was traditionally linked to worsened outcomes and exacerbated brain damage (44), other research suggests that speci c T-cell populations might actually play a protective role in brain injury recovery (45,46).Gut microbiota are known to regulate the immune cell response after TBI.
In our previous work injured mice with gut microbial depletion were found to have altered microglial morphology and increased neurodegeneration associated with impaired T-cell in ltration (17).In this manuscript, we found that in the absence of T-cells, gut-microbiota depletion had a signi cantly reduced impact on myelin repair and OLC proliferation following injury.This effect was observed both in the pharmacological deletion of CD3 + lymphocytes and in genetic absence of T-cells in TCRβ-/-TCRΔ-/-mice.These ndings provide evidence for a critical role of T-cells in gut-brain communication in the setting of TBI.Further elucidating the mechanistic and regulatory interactions between the gut microbiota, the immune response, and the brain in the setting of TBI can provide foundational knowledge for the development novel therapeutic strategies to enhance white matter repair.
While the role of resident immune cells in the CNS is well-established, the involvement of non-immune glial cells like astrocytes and OLCs in neuroin ammation has only recently emerged as a critical area of research.In the context of neurological disorders, OLCs can transition to disease-speci c cell states (47).Disease-speci c OLCs are characterized by the expression of immune speci c genes allowing the direct cross-tale between immune cell and therefore modulating immune response (35).Furthermore, defective perivascular migration of OLCs not only impairs their recruitment to the lesion site but can also disrupt the blood brain barrier, making it more permeable to in ltrating CD3 + lymphocytes (48).To further understand the role of T-cells in the modulation of OLCs we performed an in vitro co-culture with both cell types.We found that OLCs exposed to T-cells from gut-microbiota-depleted and injured mice exhibited stunted proliferation.This suppression was partially mediated by direct cell contact.Analysis of T-cells population revealed an increase in CD4 + lymphocytes expressing IL17 in gut-microbiota-depleted mice.This nding aligns with existing evidence suggesting that immune cells modulate OLC proliferation through IL17 (31,49).Furthermore, CD4 + T-cells of the Th1 and Th17 lineage play a pivotal role in MS perpetuation and establishment (50,51).Emerging evidences point to a role of Th17 cells in a wide variety of cognitive, neurovascular, and neurodegenerative diseases (52).However, additional research is required to uncover the precise mechanisms behind the gut microbial in uence on white matter repair after injury.
The overall immunomodulatory role of OLCs in CNS diseases and disorders still needs to be investigated.
Emerging evidence suggests that OLCs could modulate immune cells activation through the increased expression of gene modules associated with interferon response and MHC class I and II (35,36).These studies established a novel role of OLCs in antigen presentation in vivo (35,36,53).This novel OLCs function was further con rmed by single-nuclei RNA sequencing analysis in cortical gray matter and subcortical white matter (54).This study revealed an increased expression of MHC genes as signature of stressed oligodendrocyte in MS lesions (54).We then wondered whether T-cells isolated from injured and gut microbiota depleted animals could in uence MHC class II expression in vitro.Our data revealed that MHC-II was upregulated in OLCs in cell contact dependent manner.Furthermore, increased colocalization of MHC-II and Olig1 was observed in the peri-contusional CC after TBI with gut microbial depletion.These ndings suggest a potential mechanism by which T-cells might directly interact with OLCs through MHC-II, modifying their proliferation and amplifying their immunomodulatory role.Further research is crucial to determine whether OLCs immunomodulatory functions are prominent or merely limited to a ne-tuning effect.
There are several limitations in our investigation.Our in vivo studies included young adult mice, but the response to injury/recovery and gut dysbiosis may be in uenced by sex and age (55, 56).Furthermore, gut dysbiosis and systemic in ammation can be in uenced directly by age-related dysregulation of bile acid homeostasis (57, 58).Future research should prioritize exploring this complex interplay in the context of white matter repair.The level of antibiotics remaining in the feces of SPF mice, transplanted to GF mice, wasn't analyzed.It is possible that the GF mice had some exposure to VNAM during FMT.However, the antibiotics used are not systemically absorbed (except for metronidazole) and the second FMT was, performed 10 days before injury.Another limitation of our study is the absence of sham control in the analysis of OLCs proliferation, myelin debris accumulation and axonal swelling.Nevertheless, the proliferation of OLCs in the CC of adult uninjured mice are inherently rare under homeostatic conditions as well as the accumulation of myelin debris and axonal swelling.The absence of in vitro model of myelination and OLCs differentiation could be considered as a limitation to our study.Previous studies have identi ed accelerated remyelination in brain slice cultures with regulatory T-cells, that directly promoted OPCs differentiation (51,59).Co-staining with a marker of mature oligodendrocytes, such as proteolipid protein, or OPC speci c marker PDGFr-α would have allowed to evaluate OLCs differentiation.
The discrete area of analysis and the focal nature of the injury model are limitations to our study.Further investigation should include mild and diffuse injury models to improve the translatability to human cases of TBI.
In summary, depletion of the gut microbiota after TBI impaired OLC proliferation, increased OLC MHCII expression, and reduced white matter remyelination.Absence of T-cells protected injured mice from the detrimental effects of gut microbial depletion on WM providing evidence for T-cells as a cellular mechanistic link for gut microbiota-brain communication in the setting of TBI.Future studies should address the molecular mechanisms of gut microbial regulation of the T-cell response (such as bacterial metabolites) and its role in regulating OLC differentiation and maturation in the setting of TBI.

Declarations
Gut microbial depletion reduces oligodendrocyte lineage cell proliferation after TBI. a Experimental design: one week of VNAM after TBI; animals were sacri ced one week after injury.Figure 8

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Figure 7 Trans
Figure 7 Ethics Approval: All authors have adhered to the standards of the National Research Council's Guide for the care and use of laboratory animals and the ARRIVE guidelines.All protocols were approved by Washington University in St. Louis Animal Studies Committee.54.Schirmer L, Velmeshev D, Holmqvist S, Kaufmann M, Werneburg S, Jung D, et al.Neuronal vulnerability and multilineage diversity in multiple sclerosis.Nature.2019;573(7772):75-82. 55.Islam M, Davis BTt, Kando MJ, Mao Q, Procissi D, Weiss C, Schwulst SJ.Differential neuropathology and functional outcome after equivalent traumatic brain injury in aged versus young adult mice.Exp

Table 1 .
Overview of the antibodies for immunohistochemistry and ow cytometry used in this study Representative images of CC (white dashed lines) stained with b dMBP, c Olig1 + /BrdU + , and d bAPP.e Quanti cation of percentage of the dMBP-stained area of the CC.f Quanti cation of Olig1 + /BrdU + cells in the ipsilateral CC. g Quanti cation of bAPP axonal swelling density of the CC.h Experimental design: Germ free (GF) mice were gavaged with two fecal microbiota transplants (FMT) of the gut microbiota from mice treated with VNAM or Kool-Aid uninjured animals on Day -7 and day -17 prior to injury.i Representative uorescent images of CC (white dashed lines) stained with dMBP.j Quanti cation of percentage of the dMBP-stained area of the CC.Mean values are plotted ± SEM. *p<0.05.Unpaired t tests was used to determine statistical differences; n=5-9 mice per group.Scale bar=200 µm and 50 µm in the inserts.Abbreviations: CC: corpus callosum; CCI: controlled cortical impact; CX: cortex; dMBP: degraded myelin basic protein; FMT: fecal matter transplant; GF: germ free; SPF: speci c pathogens free; VNAM: vancomycin, neomycin-sulfate, ampicillin, and metronidazole.